Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6854—Immunoglobulins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/35—Allergens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/35—Allergens
  • A61K39/36—Allergens from pollen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/5047—Cells of the immune system
  • G01N33/505—Cells of the immune system involving T-cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• hypoallergenic hybrid molecules consisting of reassembled elements of the four grass pollen allergens which upon immunization in different animal models induced IgG antibodies blocking IgE recognition of the grass pollen allergens by allergic patients. These hypoallergenic hybrid molecules represent safe vaccines for immunotherapy of grass pollen allergy.
• IgE-mediated allergies represent a worldwide health problem with increasing prevalence (1).
• the hallmark of allergic disease is the production of IgE antibodies specific for environmental allergens, mainly from pollen, mites, animal dander, and moulds (1).
• Allergic symptoms occur, when receptor-bound IgE on mast cells or basophils is cross-linked by multivalent allergen, leading to the release of inflammatory mediators (2).
• IgE- facilitated presentation via FceRI and FceRII on antigen presenting cells strongly enhances the activation of allergen-specific T cells contributing to T cell mediated allergic inflammation (3, 4).
• Allergen-specific immunotherapy currently represents the only causative treatment of allergies with a long-term effect, although its success is impaired by the use of crude allergen extracts (5, 6).
• Linhart et al. prepared a hypoallergenic hybrid molecule with increased immunogenicity by combining hypoallergenic derivatives of the two major grass pollen allergens Phl p 2 and Phl p 6, namely a Phl p 2 mosaic molecule and a deletion mutant of Phl p 6, which was not surprising in light of previous data (Ref. 15, 17).
• the present inventors now have found that a combination of the hybrid technology and the mosaic technology does not in all cases lead to hypoallergenic molecules. Surprisingly, it has been observed that two fusion polypeptides which consisted of the same fragments but in a different order exhibited very different IgE reactivities.
• the present invention therefore provides a method for identifying polypeptides that have hypoallergenic properties and can serve as a potential vaccine.
• the present invention relates to a method for identifying hypoallergenic polypeptides, comprising the following steps: a) providing a group of polypeptides, each polypeptide comprising N fragments derived from at least two different allergens, wherein the combined amino acid sequence of any pair of two adjacent fragments within the polypeptide is not present as a consecutive amino acid sequence in any one of said allergens, wherein N is an integer greater than 3;
• At least two polypeptides within said group of polypeptides comprise the same fragments assembled in a different order.
• all fragments within said polypeptides have a length of from 20 to 100 amino acids.
• polypeptides consist of 4 to 12 fragments derived from two different allergens.
• the method further comprises the step of evaluating the secondary structure of the polypeptides provided in step (a), and selecting the polypeptide(s) which exhibit(s) random coiled structure.
• said allergens are selected from the group consisting of the grass pollen allergens Phl p 1 , Phl p 2, Phl p 3, Phl p 4, Phl p 5, Phl p 6, Phl p 7, Phl p 11 , Phl p 12 and Phl p 13.
• each fragment consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 55 through 76.
• at least one polypeptide in said group comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 through 37.
• hypoallergenic polypeptide comprising at least four fragments derived from at least two different allergens, wherein the combined amino acid sequence of any pair of two adjacent fragments within the polypeptide is not present as a consecutive amino acid sequence in said allergens, characterized in that at least one fragment is derived from Phl p 1 , Phl p 5, Phl p 2 or Phl p 6.
• hypoallergenic polypeptide comprising at least four fragments derived from at least two different allergens, wherein the combined amino acid sequence of any pair of two adjacent fragments within the polypeptide is not present as a consecutive amino acid sequence in said allergens, characterized in that each of said fragments consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 55 through 76.
• hypoallergenic polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID Nos:22, 23, 24, 25, 36 and 37.
• hypoallergenic polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs:39, 40, 41 , 42, 53 and 54.
• Another aspect of the invention is a pharmaceutical composition
• a pharmaceutical composition comprising the polypeptide of the invention and a pharmaceutically acceptable diluent or excipient.
• Another aspect of the invention is the use of the polypeptide of the invention for the manufacture of a medicament for the prevention and/or treatment of allergy, preferably of grass pollen allergy.
• Another aspect of the invention is a nucleic acid encoding the polypeptide of the present invention.
• Yet another aspect of the invention is a method of treating and/or preventing an allergic disorder, comprising administering to an individual in need thereof a therapeutically effective amount of the polypeptide or polynucleotide of this invention.
• Figure 1 depicts the design of hybrid molecules by the assembly of allergen fragments derived from the major timothy grass pollen allergens Phl p 1 , Phl p 2, Phl p 5, and Phl p 6 (see Example 1).
• Figure 2 shows a Coomassie-stained PAA-Gel containing the purified hybrid proteins A-Q (see Example 1). Molecular weights are indicated on the left margin (m, molecular weight marker).
• Figure 3 shows far UV CD spectra of the proteins B, C, P, and Q dissolved in water were collected on a Jasco J-810 spectropolarimeter (Japan Spectroscopic Co., Tokyo, Japan), see Example 2.
• Figure 4 depicts IgE-reactivity to nitrocellulose bound hybrids and control proteins for three representative grass pollen allergic patients (see Example 3).
• Figure 5 depicts reduced allergenic reactivity of hybrids in comparison to the wild-type allergens as detected by CD203c expression (see Example 4).
• Figure 6 depicts IgG reactivity after immunization of mice with a mixture of Phl p1 and Phl p 5, or a mixture of Phl p 2 and Phl p 6, or a mixture of B and C, or P, or Q.
• the development of Phl p1-, Phl p 2-, Phl p 5-, and Phl p 6-specific lgG antibody levels was compared by ELISA measurements (see Example 6).
• Figure 7 shows results from Example 7.
• the figure depicts IgG reactivity after immunization of rabbits with B, C, P, or Q, namely IgG antibody responses to the wild-type allergens Phl p 1 ( Figure 7A), Phl p 5 ( Figure 7B), Phl p 2, and Phl p 6 ( Figure 7C).
• Figure 8 depicts IgG reactivity after immunization of rabbits with B, C, P, or GPH (a grass pollen allergen-derived hybrid described in Ref. 11) namely IgG antibody responses to the wildtype allergen Phl p 1 (see Example 8).
• the method of the invention is a method for identifying hypoallergenic polypeptides.
• the method of the invention is a screening method to identifiy hypoallergenic polypeptides.
• hypoallergenic as used herein, means the reduction of IgE reactivity and ability to induce IgE-mediated mast cell or basophil degranulation.
• the method of the invention comprises providing a group of polypeptides, wherein each polypeptide within said group independently comprises N fragments derived from at least two different allergens.
• the group of polypeptides consists of at least two different polypeptides.
• the group of polypeptides consists of 2 to 100, preferably of 3 to 75, more preferably of 4 to 50, most preferably of 5 to 30 different polypeptides.
• Each polypeptide independently comprises or consists of N fragments derived from at least two different allergens.
• N is an integer greater than 3, preferably N is 4 to 25, more preferably 4 to 20, still more preferably 4 to 15, most preferably 4 to 10 (e.g. 4, 5, 6, 7, 8, 9 or 10).
• the polypeptides within the group may comprise or consist of the same or a different number of fragments. That is, N may be the same or different for the respective polypeptides within the group. Preferably, all fragments within a given polypeptide are different from each other.
• Each fragment consists of at least 8, preferably of 8 to 100, more preferably of 10 to 90, still more preferably of 12 to 80, more preferably of 15 to 70, more preferably of 20 to 60 consecutive amino acids from an allergen amino acid sequence.
• the polypeptide prepared in accordance with this invention does not necessarily consist only of amino acid sequences derived from the allergens. It is possible that non-native sequences (e.g. spacer sequences) are inserted between the fragments (which fragments are consecutive amino acid sequences from different allergens). It is also possible that the polypeptides comprise a tag sequence which facilitates the purification of the polypeptide upon expression in a host cell. An example of such a tag sequence is a hexahistidine tag which allows purification by Ni 2+ chelate chromatography. Other tags are known to those of skill in the art. Furthermore, the polypeptide may contain a foreign methionine residue at amino acid position 1 which results from expression in host cells. The methionine will often be present if the N-terminal portion of the polypeptide is an internal or C-terminal allergen fragment
• polypeptide may consist of any one of the following structures (I) to (VII):
• the polypeptide in accordance with this invention may be prepared by various methods.
• the polypeptide is prepared by expressing a polynucleotide in a host cell.
• the host cell may be a prokaryotic or eukaryotic cell. If prokaryotic cells are used the host cell is preferably E. coli. Examples of eukaryotic cells include yeast, insect cells or cell lines such as CHO cells.
• After introducing a suitable polynucleotide encoding the polypeptide of the invention into a host cell the host cell is cultured under conditions such that the polypeptide is expressed in the cell.
• the polypeptide may be secreted by the cell or accumulate inside the cell. Known purification techniques can be used to recover the polypeptide from the cell or from the culture medium.
• the polypeptide is prepared by chemical synthesis, e.g. by solid phase synthesis according to techniques that are known perse.
• allergen denotes a substance capable of eliciting a type I- hypersensitivity reaction in atopic individuals.
• IgE Immunoglobulin E
• non-parasitic antigens stimulate inappropriate IgE production, leading to type I hypersensitivity.
• allergens in the sense of the present invention include allergens from plants and animals (Allergome database: www.allergome.org).
• the allergens are usually wildtype allergens.
• the allergens may be allergens from one or more of the following species: Acarus siro, Blomia tropicalis, Dermatophagoides farinae, Dermatophagoides microceras, Dermatophagoides pteronyssinus, Euroglyphus maynei, Glycyphagus domesticus, Lepidoglyphus destructor, Tyrophagus putrescentiae, Blattella germanica, Periplaneta americana, Harmonia axyridis, Archaeopotamobius sibiriensis, Artemia franciscana, Charybdis feriatus, Crangon crangon, Homarus americanus, Litopenaeus vannamei, Metapenaeus ensis, Panulirus stimpsoni, Penaeus
• one or more allergens are allergens from the species Phleum pratense, Betula verrucosa, Dermatophagoides pteronyssinus. Most preferably, one or more allergens are allergens from the species Phleum pratense. In a preferred embodiment of the method of the invention, all allergens from which the fragments are derived are from a single species from the list recited above. That is, the different 'source allergens' are all derived from the same species.
• a preferred group of allergens in accordance with this invention are grass pollen allergens, for example allergens from the species Phleum pratensae.
• the allergens are selected from the group consisting of Phl p 1 , Phl p 2, Phl p 3, Phl p 4, Phl p 5, Phl p 6, Phl p 7, Phl p 11 , Phl p 12 and Phl p 13.
• the allergens are selected from the group consisting of Phl p 1 , Phl p 2, Phl p 5 and Phl p 6.
• the amino acid sequences of selected grass pollen allergens are shown in SEQ ID NO:1 to 10, respectively.
• the fragments are derived from at least two different allergens, preferably from 2 to 10 different allergens, more preferably from 2 to 5 different allergens, e.g. from 2, 3, 4 or 5 different allergens.
• the group of polypeptides comprises at least 2 polypeptides which consist of the same fragments but wherein the fragments are assembled in a different order.
• the method of the invention comprises determining the IgE reactivity of the polypeptides.
• IgE reactivity denotes the capability of a substance to bind to IgE antibodies. More specifically, as used herein, the phrase “IgE reactivity” refers to the capability of the polypeptide to bind to IgE antibodies from individuals that are allergic against one or more of the allergens from which the fragments within the polypeptide are derived.
• IgE reactivity may be measured by determining the degree of binding between (1) serum IgE from individuals that are allergic against one or more of the allergens from which the fragments are derived and (2) the polypeptide. This may be done by the method described in reference (18) or (19). Alternatively, IgE reactivity and allergenic activity may be determined by analysing the expression of CD203c on human basophils that were isolated from individuals allergic to one or more of said allergens. See example 4 and reference (20).
• the method of the invention comprises determining the T cell reactivity of the polypeptides.
• T cell reactivity refers to the capability of a substance to specifically bind to T cell receptors. More specifically, “T cell reactivity” means the capability of the polypeptide to induce proliferation of T cells.
• the T cell reactivity of the polypeptides can be measured by (1) providing peripheral blood mononuclear cells (PBMCs) isolated from individuals allergic against one or more of the allergens from which the fragments are derived, and (2) determining the degree of proliferation of T cells contained in said PBMCs. See example 5 and reference (16).
• PBMCs peripheral blood mononuclear cells
• the method of the invention comprises determining the capability of the polypeptides to induce an IgG response against one or more of the allergens from which the fragments are derived. This may be done by (1) immunizing a non-human mammal (e.g. a mouse, rat or rabbit) with the polypeptide, and (2) determining the amount of IgG antibodies raised in said non-human mammal, which are specific to said one or more allergen(s) from which the fragments are derived.
• the IgG antibodies measured are preferably lgG1 antibodies.
• step (2) is performed using an ELISA assay. See example 6.
• the method further comprises determining to which extent the polypeptides are capable of inducing a protective IgG response. This may be done by (1) providing a composition containing IgG antibodies by immunizing a non-human mammal (e.g. a mouse, rat or rabbit) with the polypeptide; (2) providing a composition containing IgE antibodies from individuals that are allergic against one or more of said allergens from which the fragments of the polypeptide are derived, and (3) measuring whether and/or to which extent said composition containing IgG antibodies can block the binding of said IgE antibodies to one or more of said allergens.
• a non-human mammal e.g. a mouse, rat or rabbit
• This test is preferably performed using an ELISA assay.
• the wild type allergens from which the fragments are derived may be immobilized on an ELISA plate.
• the thus pre-treated ELISA plate may then be contacted with said composition containing the IgG antibodies to allow binding of IgG antibodies to said immobilized allergens. After washing the composition containing said IgE antibodies is contacted with the ELISA plate. After washing the amount of IgE antibodies are determined. See Example 7.
• the method of the invention comprises the final step of selecting those polypeptides which exhibit favourable properties and are thus useful for the potential use as a vaccine.
• a polypeptide must have the following properties:
• the polypeptide is selected if its IgE reactivity is less than that of at least one allergen from which it is derived. Preferably, the polypeptide is selected only if its IgE reactivity is less than that of each allergen from which it is derived. For example, if the polypeptide consists of fragments derived from Phl p 2 and Phl p 5, the polypeptide must have a lower IgE reactivity than Phl p 2, and it must have a lower IgE reactivity than Phl p 5 to be selected.
• the IgE reactivity and allergenic activity are preferably reduced by at least 25%, more preferably by at least 50%, most preferably by at least 90 %, determined by quantitative IgE measurements as described in Ref. 16, and as described in Example 4.
• requirement (ii) the polypeptide is selected only if it can elicit allergen-specific T cell activation (Example 5).
• the polypeptide is selected only if it can induce an allergen- specific IgG response upon immunization (see, e.g. Example 6).
• the polypeptide is selected only if the IgG antibodies induced by immunization can inhibit allergic patients' IgE binding to the wildtype allergen (see, e.g., Example 7).
• hypoallergenic polypeptides identified by the method of the invention are hypoallergenic polypeptides identified by the method of the invention.
• the invention relates to a hypoallergenic polypeptide identified and produced in accordance with this invention.
• the hypoallergenic polypeptide may comprise or consist of at least four fragments derived from at least two different allergens, wherein the amino acid sequence of any pair of two adjacent fragments within the polypeptide is not present as a consecutive amino acid sequence in said allergens, characterized in that at least one fragment is derived from Phl p 1 or Phl p 5.
• the number of fragments may be N, wherein N has the meaning as defined above.
• the hypoallergenic polypeptide of the invention may comprise or consist of at least four fragments derived from at least two different allergens, wherein the amino acid sequence of any pair of two adjacent fragments within the fusion polypeptide is not present as a consecutive amino acid sequence in said allergens, characterized in that each of said fragments consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 55 through 76.
• SEQ ID NOs: 55 through 76 amino acid sequence selected from the group consisting of SEQ ID NOs: 55 through 76.
• the number of fragments may be N, wherein N has the meaning as defined above.
• the hypoallergenic polypeptide may consist of any one of the following structures (VIII) to (XIV):
• the tag sequence usually is 5 to 10 amino acids in length.
• hypoallergenic polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:22, 23, 24, 25, 36 and 37. These amino acid sequences are comprised in constructs B, C, D, E, P and Q, respectively (see Examples).
• the hypoallergenic polypeptide may consist of any one of the following structures (XV) to (XXI):
• the tag sequence usually is 5 to 10 amino acids in length.
• the polypeptide may consist of an amino acid sequence selected from the group consisting of SEQ ID NOs:39, 40, 41 , 42, 53 and 54.
• the constructs B, C, D, E, P and Q consist of these amino acid sequences, respectively (see Examples). These embodiments correspond to structure (VIII) or (XV) above.
• the invention further concerns a polynucleotide encoding the polypeptide of the present invention. Due to the degeneracy of the genetic code many different polynucleotide molecules may encode a single polypeptide.
• the polynucleotide of the invention preferably is an expression construct for obtaining the polypeptide after expression in host cells.
• the expression construct may further comprise components which are generally known in the art such as promoter sequences, genes encoding resistance factors against antibiotics, a replication origin and the like.
• the invention further concerns a cell transfected or transformed with a polynucleotide of the present invention.
• Suitable cells include eukaryotic cells and prokaryotic cells.
• Eukaryotic cells may be transfected by methods known in the art such as calcium phosphate mediated transfection, electroporation, lipofection etc.
• the invention further relates to a pharmaceutical composition containing the polypeptide, polynucleotide or cell according to this invention.
• the pharmaceutical composition may further contain one or more pharmaceutically acceptable carrier(s) or diluents(s) such as a buffer or salt solution.
• the pharmaceutical composition of the invention is a vaccine composition.
• the pharmaceutical composition further contains an adjuvant such as aluminium hydroxide.
• the invention also relates to a method for the preparation of the polypeptide of the invention.
• the method comprises providing a polynucleotide encoding the polypeptide, introducing said polynucleotide into a host cell, culturing the host cell thus obtained under conditions such that the hybrid polypeptide is expressed, in recovering the expression product from the cell.
• the polynucleotide may be prepared by methods known in the art. It may be preferred that PCR technology is used to prepare the polynucleotide encoding the polypeptide of the invention.
• the cDNA sequences of the grass pollen allergens Phl p 1 , 2, 3, 4, 5, 6, 7, 11 , 12, and 13 are shown in SEQ ID NO:11 to 20, respectively.
• the invention further relates to the use of the polypeptide, a polynucleotide or a cell described herein for the preparation of a medicament for the treatment and/or prevention of an allergic disorder.
• a medicament may be composed of the polynucleotide encoding a vaccine which can be used directly for the DNA-based vaccination against Type 1 allergy.
• the recombinant or synthetic polypeptide may be used to prepare formulations for the oral, sublingual or parenteral treatment of Type 1 allergic disorders as they are now routinely used for immunotherapy. Examples of formulations for sublingual immunotherapy or adjuvant bound hybrid polypeptide for injection immunotherapy.
• Possible applications include also cell-based forms of immunotherapy which may be based on e.g. dendritic cells or other antigen presenting cells. Those cells are transformed and expressed to antigen in vivo. Preferably orthologous cells transformed with suitable vectors are used.
• One mode of application may be the subcutaneous injection of adjuvant-bound polypeptide.
• Another possibility is oral or nasal administration of the polypeptide in order to induce immunological tolerance or anergy against the components of the polypeptide.
• All the possible formulations can be prepared according to measures which are known to those of skill in the art (dosage adjuvants scheme of administration).
• the invention further relates to the use of the polypeptide described herein or of a polypeptide or a cell described herein for the preparation of a medicament for prophylactic vaccination or tolerance induction.
• Prophylactic administration of hybrid polypeptides means the administration of the polypeptide to individuals, preferably children who do not yet suffer from Type 1 allergy in order to induce a state of immunological tolerance, anergy or non-responsiveness, or a protective immunity against the components of the hybrid vaccine. This may be achieved by the various protocols outlined for treatment of an established allergic disorder.
• the prophylactic treatment may be performed with the polypeptides or polynucleotides described herein above.
• the invention relates to the use of a polypeptide described herein for the detection of antibodies against an allergenic protein in a sample.
• the antibody may be an IgM IgE, IgG or IgA antibody.
• the concentration of the antibody may be determined from a sample which has been obtained from a body fluid.
• the sample may be derived from animals or humans.
• Such tests may rely on a solid phase immobilized polypeptide or the polypeptide in the fluid phase. Examples for such tests include ELISA tests, Western blotting tests or any other tests where the polypeptide is immobilized to bind to specific antibodies out from the sample.
• the polypeptide is added directly to the antibody containing fluid in order to adsorb specific antibodies as, e.g., in competitive immunological assays.
• the polypeptide of the invention may also be used for cellular tests such as a T cell proliferation test, etc.
• amino acid sequences SEQ ID NO: 1-10 show the mature peptides lacking the signal peptide, where applicable.
• hypoallergenic hybrid molecules seventeen different hybrid molecules were designed by the assembly of allergen fragments derived from the major timothy grass pollen allergens Phl p 1 , Phl p 2, Phl p 5, and Phl p 6 as shown in Figure 1.
• the amino acid sequences of the resulting proteins are listed in Table 1.
• P1 M and P2M refer to previously designed allergen derivatives (References 15, 16). All sequences were codon-optimized for expression in Escherichia coli, a start-codon (ATG) was added at the 5' end and a 6xHistidine tag was added at the 3'end of each sequence, followed by a stop-codon.
• Example 4 Reduced allergenic activity of the hybrid molecules B, C, P, and Q
• CD203c has previously been described as an activation marker on human basophils, which is upregulated upon allergen-induced cross-linking of receptor-bound IgE (20).
• the cells tolerated an at least 10 fold higher concentration of the hybrid molecules compared to the equimolar amount of the wildtype allergens (Patients 1-12).
• Example 6 Immunization with the hybrid molecules B, C, P, and Q induced an IgG response directed against the wildtype allergens
• the hybrids B+C, as well as P and Q were able to induce a Phl p 1 , Phl p 2, Phl p 5, and Phl p 6-specific IgGi antibody response, which were higher than the antibody response induced by the wildtype allergens themselves.
• Example 8 Immunization with the hybrid molecules B. C, and P induced an IgG response directed against the wildtyp allergen Phl p 1
• Combination vaccines for the treatment of grass pollen allergy consisting of genetically engineered hybrid molecules with increased immunogenicity. FASEB J. 16, 1301-1303. Linhart, B., Haiti, A., Jahn-Schmid, B., Verdino, P., Keller, W., Krauth, M.T., Valent, P., Horak, F., Wiedermann, U., Thalhammer, J., Ebner, C, Kraft, D., and Valenta, R. (2005).
• Swoboda I Bugajska-Schretter A, Linhart B, Verdino P, Keller W, Schulmeister U, Sperr WR, Valent P, Peltre G, Quirce S, Douladiris N, Papadopoulos NG, Valenta R, Spitzauer S. A recombinant hypoallergenic par albumin mutant for immunotherapy of IgE- mediated fish allergy. J Immunol. 2007.178:6290-6.
• a hypoallergenic hybrid molecule with increased immunogenicity consisting of derivatives of the major grass pollen allergens, Phl p 2 and Phl p 6. Biol. Chem. 389:925- 33.

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